Recombinant P. multocida Hyaluronan Synthase/HAS Protein, CF

Catalog #: 9585-GT Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
9585-GT-050

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Recombinant P. multocida Hyaluronan Synthase/HAS Protein, CF Summary

Learn more about Fluorescent Glycan Labeling and Detection

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to transfer GlcNAc and GlcA from donor substrates to hyaluronan. The specific activity is >80 pmol/min/μg, as measured under the described conditions.

Source

E. coli-derived p. multocida Hyaluronan Synthase protein
Met1-Ile703, with C-terminal 6-His tag

Accession #

Q7BLV3

N-terminal Sequence
Analysis

Met1

Predicted Molecular Mass

81 kDa

SDS-PAGE

74 kDa, reducing conditions

Product Datasheets

9585-GT

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

9585-GT

Formulation	Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Glycosyltransferase Activity Kit (Catalog # EA001)
10X Assay Buffer (supplied in kit): 250 mM Tris, 100 mM CaCl₂, pH 7.5
MnCl₂ (supplied in kit): 100 mM
Recombinant P. multocida Hyaluronan Synthase (rP.multocida HAS) (Catalog # 9585-GT)
UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
UDP-GlcA (Sigma, Catalog # U5625), 10 mM stock in deionized water
Hyaluronan (Ultra-Low MW) (Catalog # GLR003), 100 mg/mL in deionized water
96-well Clear Plate (Catalog # DY990)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Prepare 1X Assay Buffer containing 10 mM MnCl₂ by combining 10X stocks and diluting 10 fold with deionized water.
Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
Prepare reaction mixture containing 0.4 mM UDP-GlcNAc, 0.4 mM UDP-GlcA, 8 mg/mL Hyaluronan, and 4 µg/mL Coupling Phosphatase 1 in 1X Assay Buffer.
Dilute rP.multocida HAS to 16 ng/µL in 1X Assay Buffer.
Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
Load 25 µL of 16 ng/µL rP.multocida HAS into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
Seal plate and incubate at 37 °C for 20 minutes.
Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
Add 100 µL of deionized water to all wells. Mix briefly.
Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
Read plate at 620 nm (absorbance) in endpoint mode.
Calculate specific activity:

Specific Activity (pmol/min/µg) =	Phosphate released* (nmol) x (1000 pmol/nmol)
	Incubation time (min) x amount of enzyme (µg)

*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

Per Reaction:

rP.multocida HAS: 0.4 µg
Coupling Phosphatase 1: 0.1 µg
UDP-GlcNAc: 0.2 mM
UDP-GlcA: 0.2 mM
Hyaluronan: 200 µg

Reconstitution Calculator

Background: Hyaluronan Synthase

Hyaluronan (HA) is a polysaccharide chain composed of repeating beta 4GlcUA-beta 3GlcNAc disaccharide units with molecular weights generally ranging from ~10⁴ to 10⁷ Da in vertebrates and bacteria (1, 2). In animals, HA plays structural, recognition and signaling roles. Certain pathogenic bacteria, namely Streptococcus Group A and C and Pasteurella multocida Type A, utilize extracellular HA polysaccharide capsules to avoid host defenses and to increase virulence. It is now recognized that HA of different sizes can have dramatically different effects on cellular behavior and growth (3, 4), and vertebrates may be able to control HA size in vivo by differential expression of biosynthetic enzymes (5). The Pasteurella multocida HA synthase enzyme, pmHAS, catalyzes the synthesis of HA polymer by alternative addition of GlcNAc and GlcA residues to the nonreducing terminus of HA using the donor substrates UDP-GlcNAc and UDP-GlcA (6). The enzyme can also elongate exogenous HA oligosaccharide acceptors in vitro (7, 8), therefore can be used for non-reducing end labeling of HA. The enzymatic activity of pmHAS was determined using a phosphatase-coupled assay (9).

References